Tetrahymena thermophila is a very useful microbial organism for fundamental eukaryotic experimental cell and molecular biology and for comparative genomics. It is a free-living, fresh-water unicellular organism, which has conserved a fairly complete set of ancestral eukaryotic animal biology. These features make it an alternative eukaryotic microbe with excellent credentials for fundamental cell and molecular investigation of human biology that has been evolutionarily lost by yeast. Tetrahymena's germline (micronuclear) genome consists of five pairs of chromosomes. Its somatic or expressed (macronuclear) genome consists of approximately 300 chromosomes, generated by site-specific fragmentation of the germline genome. The goal of this project is to make comprehensive and robust HAPPY physical maps of both genomes, based on STSs spaced 50 kb apart. 4,000 expressed sequence tags (ESTs) will constitute the backbone of the physical map. The map will also include two other types of STS: 1) chromosome breakage junctions, to serve as the well defined ends to the physical map of each macronuclear chromosome fragment and to allow the ordered concatenation of the physical maps of individual macronuclear chromosomes into a physical map of the entire germline genome and 2) cloned RAPD STS, which in conjunction with polymorphic chromosome breakage junctions, will be used to extensively link the physical map to the genetic maps. When the aims of this proposal are completed, we expect to have: a) The first physical count of the number of MAC chromosomes in the Tetrahymena genome and of their relative copy numbers. b) A physical map of every Tetrahymena MAC chromosome, linked from one telomere to the other, with STS markers spaced every 50 kb. c) A physical map of the MIC genome that includes the location of all the chromosome breakage junctions and which accurately reflects the order and orientation of every MAC chromosome. d) A physical map that includes greater than 200 polymorphic STS, providing robust links of the physical map to our relational MIC:MAC genetic maps and eventually to the sequence map. e) ESTs, representing 10-20 percent of the estimated total number of genes in Tetrahymena and contributing a wealth of comparative genomics information of functional and evolutionary significance. f) The likelihood of continuing to generate unexpected and valuable fundamental knowledge about the cell, genetic and molecular biology of this important organism.